Detection of Enrofloxacin After Single-Dose Percutaneous Administration in Python regius, Boa constrictor imperator, and Acrantophis dumerili

Detection of Enrofloxacin After Single-Dose Percutaneous Administration in Python regius, Boa constrictor imperator, and Acrantophis dumerili

Author’s Accepted Manuscript DETECTION OF ENROFLOXACIN AFTER SINGLE-DOSE PERCUTANEOUS ADMINISTRATION IN PYTHON REGIUS, BOA CONSTRICTOR IMPERATOR, AND ...

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Author’s Accepted Manuscript DETECTION OF ENROFLOXACIN AFTER SINGLE-DOSE PERCUTANEOUS ADMINISTRATION IN PYTHON REGIUS, BOA CONSTRICTOR IMPERATOR, AND ACRANTOPHIS DUMERILI Alban Ducrotte-Tassel, Plamen Kirilov, Jean-Paul Salvi, Iga Czyz, Vanessa Doré, Geneviève Marignac, Charles-Pierre Pignon, Roselyne Boulieu, Sébastien Perrot

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S1557-5063(17)30208-2 http://dx.doi.org/10.1053/j.jepm.2017.08.002 JEPM744

To appear in: Journal of Exotic Pet Medicine Cite this article as: Alban Ducrotte-Tassel, Plamen Kirilov, Jean-Paul Salvi, Iga Czyz, Vanessa Doré, Geneviève Marignac, Charles-Pierre Pignon, Roselyne Boulieu and Sébastien Perrot, DETECTION OF ENROFLOXACIN AFTER SINGLE-DOSE PERCUTANEOUS ADMINISTRATION IN PYTHON REGIUS, BOA CONSTRICTOR IMPERATOR, AND ACRANTOPHIS DUMERILI, Journal of Exotic Pet Medicine, http://dx.doi.org/10.1053/j.jepm.2017.08.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Research

Detection of Enrofloxacin After Single-Dose Percutaneous Administration in Python regius, Boa constrictor imperator, and Acrantophis dumerili Alban Ducrotte-Tassel, DVM, MS Plamen Kirilov, MS, PhD Jean-Paul Salvi, PhD Iga Czyz, DVM, MS Vanessa Doré, MS Geneviève Marignac, DVM, MS, PhD Charles-Pierre Pignon, DVM, MS Roselyne Boulieu, Pharm D, PhD Sébastien Perrot, Pharm D, PhD From the Université Claude Bernard Lyon 1, Biologie Tissulaire et Ingénierie Thérapeutique UMR 5305 – Vecteurs Colloïdaux et Transport Tissulaire, Institut des Sciences Pharmaceutiques et Biologiques, Lyon, France (Ducrotte-Tassel, Kirilov, Salvi, Czyz, Boulieu), Université ParisEst, Ecole Nationale Vétérinaire d’Alfort, Maisons-Alfort, France (Doré, Marignac, Pignon, Perrot), CHU de Lyon, Unité de Pharmacocinétique Clinique, Lyon, France (Boulieu). Address correspondence to Plamen Kirilov, MS, PhD, Université Claude Bernard Lyon 1, Biologie Tissulaire et Ingénierie Thérapeutique UMR 5305 – Vecteurs Colloïdaux et Transport Tissulaire, Institut des Sciences Pharmaceutiques et Biologiques, 8 avenue Rockefeller, 69373 Lyon cedex 08, France. Email address: [email protected] Tel: +33-4-7877-7113. Fax: +33-4-7877-7247

Abstract In this study, the blood concentration of enrofloxacin administered transdermally to three different reptilian species, Python regius, Acrantophis dumerili, and Boa constrictor imperator at 50 mg/kg was determined. The formulation used was a transdermal commercial vehicle, Pentravan® cream, a hydrophilic base emulsion that uses the liposomal technique to penetrate the skin. The enrofloxacin was incorporated at 5 wt % in the Pentravan® cream, and the plasma level of enrofloxacin and its metabolite ciprofloxacin were measured using high-performance liquid chromatography (HPLC). The results showed that the detected amounts of enrofloxacin and ciprofloxacin (0.33 and < 0.15 µg/mL, respectively) were below the limit of quantification (LOQ). Enrofloxacin was detected in P. regius and B. constrictor while ciprofloxacin was detected only in A. dumerili. Although the values were unquantifiable, this study confirms the resorption of enrofloxacin. Therefore these findings suggests that enrofloxacin may be a candidate for treatment using the transdermal route in certain reptile species. Key words: enrofloxacin; reptiles; transdermal administration; Pentravan® cream; HPLC

Enrofloxacin (1-cyclopropyl-6-fluoro-7-[4-ethyl-1-piperazinyl]-1,4-dihydro-4-oxo-3quinolinecarboxylic acid), belongs to the fluoroquinolone family of compounds, which is a subfamily of quinolone (Fig. 1). The first quinolone, nalidixic acid, was used in animals in the early 1980s while enrofloxacin was the first fluoroquinolone patented in 1984.1 The major advancement in the quinolone family was the addition of a fluorine atom at the sixth position, which improved quinolone’s antibacterial spectrum2, created the fluoroquinolone subfamily with higher lipophilic properties and consequently improved transdermal absorption.3, 4 Enrofloxacin is effective against a variety of gram-negative bacilli and cocci, particularly the Enterobacteriaceae. This antibiotic compound has therapeutic activity against gram-positive aerobes and demonstrates activity against other organisms, including Chlamydia, Rickettsia, Mycoplasma, and Mycobacterium species.5,6 The pharmacokinetics of enrofloxacin have been investigated in several reptile species including Python molurus, Iguana iguana, and Crocodilus porosus.7,8,9 These studies used intramuscular (IM), intravenous (IV), or per os (PO) routes to administer the enrofloxacin. Local irritation due to intramuscular administration has been reported and drug absorption by reptiles, following oral administration, has been documented.10-15 Despite its potential complications, enrofloxacin is still used in reptiles because of its effectiveness against a wide variety of gram-negative and mycoplasma pathogens and may be indicated for treating infectious diseases (e.g., pneumonia, stomatitis, abscess, meningitis).16 In practice, enrofloxacin is used at 5 – 10 mg/kg once a day for 10–30 days longer for reptile species, depending on the severity of the disease diagnosed.17,18 The objective of this study was to characterize the pharmacokinetics of a single transdermal dose of enrofloxacin and its metabolite, ciprofloxacin (Fig. 1).

Pentravan®, a transdermal enhancer, was administered to three species of reptiles commonly kept as pets in France (Python regius, Acrantophis dumerili, and Boa constrictor imperator) to test the subsequent hypothesis: if 5 wt % of enrofloxacin in Pentravan vehicle is systemically absorbed by transdermal administration in Python regius, Acrantophis dumerili, and Boa constrictor imperator, then enrofloxacin or its active metabolite, ciprofloxacin, should be detected in blood samples following administration.

MATERIALS AND METHODS Chemicals The enrofloxacin and ciprofloxacin hydrochloride were provided by Fagron (Rotterdam, Netherlands) while the gradient grade methanol and phosphoric acid (85 %) were purchased from Merck (Rahway, NJ, USA). The gradient grade acetonitrile and hydrochloric acid were purchased from VWR® (Fontenay Sous Bois, France) and perchloric acid (70 %) from Sigma-Aldrich (St. Louis, MO, USA). The deionized water was obtained by purification using a water purification system Purelab Option (ELGA LabWater, High Wycombe, UK). Pentravan® Cream Base is an oil-in-water emulsion with a liposomal matrix that uses the same penetration enhancing ingredients as Pluronic Lecithin Organogel (PLO) which enables it to establish a greater rate and extent of absorption of the drug than other transdermal bases. 19 Moreover, Pentravan® Cream Base is a true vanishing cream, leaving no sticky residue and providing a cosmetically elegant skin feel. Therefore, there is no need to cover the area of application to prevent transferring of the cream and to ensure effectiveness of

therapy. It is preservative and fragrance-free. Pentravan® cream composition and the functional category of its ingredients are reported in Table 1. Animals The investigation was performed on nine healthy specimens from three different species of ophidians, P. regius, A. dumerili, and B. constrictor from a zoo located near Paris, France (Fig. 2). The animals were kept in reptile breeding racks equipped with Néon UVB 2.0 light sources at controlled temperature (26 – 30°C) and humidity (40 – 50 %). The animals were fed rats weekly (1 or 2 rats weighing between 200 and 500 g each) while water was available in the enclosures. The ophidians were weighed to determine the amount of blood that could be collected (Table 2). During the experimental period, the animals were housed using the environmental parameters appropriate for each species. Young adults and juveniles were used in this study. At the end of the study, no abnormal behavior or physical abnormalities was reported in the nine subject animals. All the species used are listed under Appendix II of the Convention on International Trade in Endangered Species (CITES) except A. dumerili, which is listed under Appendix I. The investigation was conducted under the guidelines of the National Charter on Ethics of Animal Experimentation published by the National Committee for Ethics Reflection in Animal Experimentation in January 2008, and with the consent of the owners of the animals.

Transdermal formulation and treatment procedure The formulation used in this study was Pentravan®, a hydrophilic base emulsion that uses the liposomal technique to penetrate the different skin layers and cells. The mixture was prepared in the pharmacy of the Veterinary School of Alfort using the

best practices veterinary extemporaneous preparations. For the experimental preparation 250 mg of enrofloxacin was dispersed into 50 g of Pentravan® cream, which was then sealed in a pot. The homogeneity of the enrofloxacin distribution and stability were checked through the use of optical microscopy.20 After the animals were weighed the formulation was then manually applied directly to the nape of the neck using rubber gloves. The site of application was selected since injections are commonly administered in the cranial third of the body because reptiles possess a renal portal system that may shunt administered drugs to the last third of the body.21 The enrofloxacin dosage was adjusted according to results obtained in the authors’ previous in-vitro absorption study that indicated 10 % of the active ingredient passed through the shed model membrane.33 Consequently, for this study the formulation contained 5 mg/kg of enrofloxacin for a dosage of 1 g formulation per 1 kg of snake. After the enrofloxacin formulation had been applied, blood samples were collected. A blood sample was collected from each animal by cardiocentesis before the application of the formulation (t = 0) via a 25G needle attached to a syringe. Subsequent blood samples were collected from the same site of each animal (Fig. 3) then placed into 1 ml vacutainer EDTA tubes (BD Microtainer). Each blood sample collected was less than 1 % of the animal body weight or 1 mL/100 g per animal to avoid inducing anemia following multiple blood withdrawals (Table 3).

Experimental protocol For the pharmacokinetic study, the blood samples were collected at 0, 1, 2, 4, 24, 48, 72, and 96 hours after application of the 5 wt % enrofloxacin formulation. Then, the samples were centrifuged at 5000 rpm for 5 min immediately after cardiocentesis, the

plasma was transferred in Eppendorf tubes, frozen at -20 °C, and shipped on dry ice to our laboratory for analysis. Protocol to determine enrofloxacin concentration Enrofloxacin and its active metabolite, ciprofloxacin were analyzed using a high-performance liquid chromatography (HPLC) system (Waters Alliance 2795 with a Waters 2996 ultraviolet (UV) detector DAD and Computer Software Waters Empower). The column used was a Kinetex 5-µm C18 100A, size 150 × 4.6 mm maintained at 25 °C. The mobile phase consisted of 0.002 M phosphoric acid/acetonitrile (83/17, v/v). The calibration curves were linear in the range of 1–100 µmol/L for enrofloxacin and ciprofloxacin. The limit of quantification (LOQ) of enrofloxacin and ciprofloxacin was 2 and 3 µmol/L (0.7 and 1 µg/mL) respectively, while the limit of detection (LOD) for both compounds was 0.5 µmol/L (0.13 µg/mL).

RESULTS At the doses used in this study (5 mg/kg) and at each time point investigated (0, 1, 2, 4, 24, 48, 72, and 96 h), the amounts of enrofloxacin and ciprofloxacin (0.33 µg/mL and < 0.15 µg/mL, respectively) detected were below the LOQ (Fig. 4). Although the values were not quantifiable, enrofloxacin and ciprofloxacin were detectable demonstrating they crossed the reptiles’ skin barrier. Enrofloxacin was detected in all species while ciprofloxacin was detected in all A. dumerili subject animals and 2 of 3 P. regius and B. constrictor imperator.

DISCUSSION Previous studies have focused on the pharmacokinetics of enrofloxacin following intramuscular or oral administration.5-10, 23 Enrofloxacin was transdermally administered in this experiment because this route has not been conventionally used in reptiles. This new route was investigated because it can facilitate the administration of antibiotics and limit the side effects associated with parenteral administration of enrofloxacin, including tissue necrosis, discoloration, and pain. To the best of the authors’ knowledge, this is the first in vivo study to demonstrate the transdermal penetration of snakeskin by enrofloxacin. The detected concentrations of enrofloxacin were below the LOQ. The enrofloxacin concentrations being below the LOQ could be related to the influence of several factors affecting the transdermal absorption such as the temperature at which the snakes were kept, their phase of the shed cycle, their age and sex, the site at which the medication was applied, the vascularity of the underlying tissues, the properties of the Pentravan vehicle, the concentration of enrofloxacin, or the dose that was applied. In a previous study on the royal python (P. regius, n = 6), 39 blood samples were collected by cardiocentesis from each animal over a period of 120 days. No clinical complications were reported in any of the six pythons used in the experiment up to 73 days after the last blood samples were collected.24 Therefore, cardiocentesis was chosen as the method of blood sample collection. In our study, the reptiles were properly maintained, and the samples were collected by a competent and experienced handler to ensure that the animals did have any adverse effects associated with the procedure. The renal portal system controls the volume of blood that flows to the kidneys. By allowing venous blood to pass through the kidneys, the risk of ischemia due to

decreased or impaired perfusion is reduced. Furthermore, it is assumed that more blood passes through the portal system in cases of water deprivation in desert species.21 Currently, the mechanisms involved in the renal portal system are not known but in the red-eared slider (Trachemys scripta elegans), a valve has been identified as a structure that regulates the direction of blood flow to the kidneys.25 Recent studies on turtles and pythons have failed to demonstrate a significant effect of the injection site on the pharmacokinetics of drugs, even in those where the kinetics should be altered by the renal portal system.22 However, it is believed that no alterations are induced in the pharmacokinetics of the renal portal system following cranial administration to the body. In our previous ex-vivo study the authors’ showed that the transcutaneous passage of enrofloxacin was better in the cranial part of the body close to the head than across the skin sampled close to the cloaca.26 Therefore, the ointment was applied to the first third of the body of the snakes. This study was conducted in a room where the temperature varied between 26 and 30°C, corresponding to the optimal temperature range of the three reptile species used in this investigation. Poikilotherms require a certain temperature to ensure that their metabolism is efficient, which is even more important for animals undergoing treatment. Several previous pharmacokinetic studies focused on developing a single application dose and a route of administration of enrofloxacin.27-30 For example, the absorption and tissue distribution of enrofloxacin and of its main metabolite ciprofloxacin were investigated in ducks after oral or intramuscular administration of a single dose of 10 mg/kg enrofloxacin.27 The peak concentrations of enrofloxacin after intramuscular administration (1.67 µg/mL at 0.9 h) were higher than after an oral dose (0.99 µg/mL at 1.38 h). It was concluded that a dose of 10 mg/kg per day

provides serum and tissue concentrations sufficiently high to be effective in the control of many bacterial infections diagnosed in ducks. Concerning transdermal administration, tissue concentrations of enrofloxacin and ciprofloxacin were determined over a 24 hour period after a single transdermal application dose of enrofloxacin (10 mg/kg) in the coqui frog (Eleutherodactylus coqui).28 Enrofloxacin tissue concentrations ranged from 0.2 to 0.44 μL/mL, whereas ciprofloxacin concentrations ranged from 0.42 to 0.81 μL/mL.28 In the study described in this report, the formulation was also applied only once to the snakes, but only a 5 mg/kg dose was used. To further investigate this new route of administration, other studies should be conducted using repeated drug application to determine potential cumulative effects. The alternative option would be to prepare a more concentrated formulation (10 or 20 wt %) with an appropriate galenical preparation. Pentravan® cream was used in this study based on human clinical formulation for extemporaneous medicinal preparations that are applied to the skin. If the transition is shown to be less than optimal, it would be interesting to attempt to discover another transdermal base, including other excipients. In a previous ex vivo transdermal study, the use of an organogel allowed an obviously superior passage of enrofloxacin when a porcine skin sample was used as a membrane model in a Franz cell diffusion apparatus.22 This present study demonstrates the feasibility of organogel formulations as potential drug delivery systems, which could be used for transdermal drug administration in reptiles.

CONCLUSION The present study suggests that enrofloxacin can be systemically absorbed by the three snake species used as subject animals when applied in a Pentravan vehicle,

but its therapeutic efficacy could not be quantified. In addition, the authors’ revealed that a single application of 5 wt % enrofloxacin ointment in Pentravan vehicle did not provide quantifiable concentrations in blood for 96 hours’ post-administration under certain environmental conditions. This study sought to demonstrate a new route of administration in reptiles. To validate this method, future studies should investigate the effects of different vehicles, different concentrations or formulations of cream, different sites of administration, different ambient temperatures, and different frequencies and durations of administration on plasma drug levels. Finally, transdermal administration of therapeutics for reptiles can have major benefits for companion exotic animals, reptile production facilities, or large captive collections. Therefore, it would be expedient to perform further studies to confirm the results obtained here and provide additional valuable information.

ACKNOWLEDGEMENTS The authors gratefully acknowledge all the people working in the Laboratory of Industrial Pharmaceutics Technologies of University of Lyon 1. In addition, we would like to thank Charlotte Hubler, the manager of the Acclimatization Center of "La Ferme Tropicale" of Combs-la-ville for all the time spent assisting with the blood sampling.

REFERENCES 1. Grohe K, Zeiler H-J, Metzger KG: 7-amino-1-cyclopropyl-4-oxo-1, 4-dihydroquinoline-and naphthyridine-3-carboxylic acids and antibacterial agents containing these compounds. US4670444 (A) 1987 2. Wright DH, Brown GH, Peterson ML, et al: Application of fluoroquinolone pharmacodynamics. J Antimicrob Chemother 46(5):669-683, 2000 3. Allen LV Jr: Transdermals: the skin as part of a drug delivery system. Int J Pharm Compd 15(4):308-315, 2011 4. Piddock LJV, Jin YF, Griggs DJ: Effect of hydrophobicity and molecular mass on the accumulation of fluoroquinolones by Staphylococcus aureus. J Antimic Chemother 47:261-270, 2011 5. Prescott JF, Yielding KM: In-vitro susceptibility of selected veterinary bacterial pathogens to ciprofloxacin, enrofloxacin and norfloxacin. Can J Vet Res 54:195-197, 1990 6. Walker RD: Fluoroquinolones, in: Prescott JF, Baggot JD, Walker RD (eds.): Antimicrobial therapy in veterinary medicine, 3rd ed. Ames, IA, Iowa State University Press, pp. 315-338, 2000 7. Martelli P, Lai OR, Krishnasamy K: Pharmacokinetics behavior of enrofloxacin in estuarine crocodile (Crocodylus porosus) after single intravenous, intramuscular and oral doses. J Zoo Wild Med 40:696-704, 2009 8. Maxwell LK, Jacobson ER: Preliminary single-dose pharmacokinetics of enrofloxacin after oral and intramuscular administration in green iguanas (Iguana iguana). Proc Am Assoc Zoo Vet 25:89-92, 1997

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17. Rota MS, Giorgi T, Capasso M, Briganti A: Blood concentrations of enrofloxacin and the metabolite ciprofloxacin in yellow-bellied slider turtles (trachemys scripta scripta) after a single intracoelomic injection of enrofloxacin. J Exot Pet Med 22(2):192-199, 2013 18. Kaplan’s M: Ulcerative stomatitis (mouthrot) in reptiles. J Wildlife Rehab 18(2):13-19, 1995 19. Allen LV Jr: Transdermals: the skin as part of a drug delivery system. Int J Pharm Compd 15(4):308-315, 2011 20. Lafargue ME, Biogeaud S, Rutledge DN, et al: Proficiency testing schemes: solutions for homogeneity control. Accred Qual Assur 9: 333- 339, 2004 21. Holz P, Barker IK, Crawshaw G.J, et al: The anatomy and perfusion of the renal portal system in the red-eared slider (Trachemys scripla elegans). J Zoo Wildl Med 28:378-385, 1997 22. Raphael BL, Papich M, Cook R.A: Pharmacokinetics of enrofloxacin after single intramuscular injection in Indian star tortoises (Geochelone elegans). J Zoo Wildl Med 25:88-94, 1994 23. Wang JC: Moving one DNA double helix through another by a type II DNA topoisomerase: the story of a simple molecular machine. Q Rev Biophys 31:107-144, 1998 24. Holz PH, Raidal SR: Comparative renal anatomy of exotic species. Vet Clin North Am Exot Anim 9:1-1, 2006 25. Kirilov P, Van Tran H, Ducrotté-Tassel A, et al: Ex-vivo percutaneous absorption of enrofloxacin: Comparison of LMOG organogel vs. pentravan cream. Int J Pharm 498(1-2):170-177, 2016

26. Ducrotté-Tassel A, Kirilov P, Salvi JP, et al: Ex-vivo permeation of enrofloxacin through shed skin of Python molurus bivittatus, as evaluated with a Franz cell. J Drug Deliv Sci Tec 36:89-94, 2016 27. Intorre L, Mengozzi G, Bertini S, et al: The plasma kinetics and tissue distribution of enrofloxacin and its metabolite ciprofloxacin in the muscovy duck. Vet Res Commun 21(2):127-136, 1997 28. Knoll U, Glünder G, Kietzmann M: Comparative study of the plasma pharmacokinetics and tissue concentrations of danofloxacin and enrofloxacin in broiler chickens. J Vet Pharmacol Ther 22(4):239-246, 1999 29. Boothe M, Boeckh A, Boothe WH, et al: Tissue concentrations of enrofloxacin and ciprofloxacin in anesthetized dogs following single intravenous administration. Vet Ther 2(2):120-128, 2001 30. Valitutto MT, Bonnie LR, Calle PP, et al: Tissue concentrations of enrofloxacin and its metabolite ciprofloxacin after a single topical dose in the coqui frog (Eleutherodactylus coqui). J Herpet Med Surg 23(3-4):69-73, 2013

Table 1. Pentravan® cream ingredients and their functional categories

Table 2. Characteristics of ophidians used in study

Table 3. Enrofloxacin and ciprofloxacin amounts detected in snake specimens

FIGURE LEGENDS Figure 1. Fluoroquinolone chemical structures (a) nalidixic acid, (b) ciprofloxacin, and (c) enrofloxacin. Figure 2. Studied ophidian species (a) Python regius, (b) Acrantophis dumerili, and (c) Boa constrictor imperator. Figure 3. Blood sample collection from a ball python (Python regius) using cardiocentesis. Figure 4. Chromatogram of standards solutions spiked with ciprofloxacin and enrofloxacin at a concentration of 50 µM.